J Mol Evol 2002) 55:364±374 DOI: 10.1007/s00239-002-2337-x Molecular Phylogeny of the Extinct Pleistocene Dwarf Elephant Palaeoloxodon antiquus falconeri from Tilos Island, Dodekanisa, Greece Nikos Poulakakis,1 Georgios E.Theodorou, 2 Eleftherios Zouros,3 Moysis Mylonas1 1 Natural History Museum of Crete NHMC), University of Crete, P.O. Box 2208, GR 71409, Irakleio, Crete, Greece 2 Subfaculty of Earth Sciences, Department of Historical Geology and Palaeontology, University of Athens, Athens, Greece 3 Institute of Marine Biology of Crete IMBC), P.O. Box 2214, GR 71003, Irakleio, Crete, Greece Abstract. A partial sequence of cytochrome b 228 1996), fossil DNA has not been used for the resolu- bp) gene of mitochondrial DNA was successfully tion of systematics problems, such as the problems of determined from rib bones of the dwarf elephant the elephantine taxa. Palaeoloxodon antiquus falconeri BUSK, which were Maglio 1973) has suggested that the immediate excavated from Charkadio cave of the island of Tilos, ancestry of the Elephantidae must be sought among Dodekanisa, Greece. This is the ®rst report of DNA the gomphotheres of Africa Primelephas gompho- sequence of a dwarf elephant. The sequences were theroides) before the early Pliocene, approximately used to examine the phylogenetic relationships ®ve million years ago Fig. 1). Based on stratigraphic among Elephantidae. Phylogenetic trees reconstruc- data, three lineages of elephantine proboscideans had ted by the neighbor-joining and maximum parsimony become established. The ®rst group, which includes methods provided identical topologies. The results the living African elephant Loxodonta africana)and support the ``Palaeoloxodon±Elephas'' clade, which is its congeners, originated probably in sub-Saharian consistent with previous morphological reports ac- Africa and expanded throughout of the continent cording to which Palaeoloxodon is more closely re- during the Middle Pleistocene. The second phyletic lated to Elephas than to Loxodonta or Mammuthus. group, which includes the species of the genus Mammuthus, originated in sub-Saharian Africa and Key words: Dwarf elephant Ð Palaeoloxodon an- had a rapid expansion northward, becoming extinct tiquus falconeri Ð Insularity Ð Quaternary Ð Tilos in its former range. In the early Villafranchian age Ð Greece Ð mtDNA Ð Molecular phylogeny Ð around 2 mya), Mammuthus spread into southern Elephant evolution Ð Ancient-DNA Ð cyt b Europe. European access in the latest Pliocene was followed by expansion into north Asia and North America at the end of early Pleistocene and became Introduction extinct in Holocene 10,000 years ago). The third phyletic group includes the living Asian elephant Ancient DNA retrieved from postmortem bones and Elephas maximus) and congeners. They originated in soft tissues provide scientists with powerful molecular sub-Saharian Africa, as the other two groups that evidence to examine the genetic changes and phylo- mentioned before and have a rapid expansion into genetic relationships between extant and extinct taxa, eastern and southern areas. Only later ranged into even when morphological and anatomical informa- North Africa. An early expansion into Asia via Aden tion is limited. Unfortunately, with few exceptions land bridge in late Pliocene time resulted in a radia- HaÈ nni et al. 1994; DeSalle et al. 1992; Yang et al. tion of forms in the physiographically complex southern Asiatic region. A second expansion from Correspondence to: Nikos Poulakakis; email: paleont@nhmc. Africa probably via Middle East during the closing voc.gr phases of the early Pleistocene moved from here into 365 Fig.1. The phylogenetic relationships between all species of the family Elephantidae Maglio 1973). Some modi®cations made by us. The 3 geological timescale is given on the right mya = million years ago). Europe and Asia during the Middle Pleistocene. The dioimmunoassays were able to determine Elephas, pygmy elephants of Sicily, Malta, Crete, and other Loxodonta, and Mammuthus as closely related taxa, islands of the Mediterranean area were distinct from but could not resolve the relationships within the Elephas antiquus in a number of important features, subfamily Lowenstein et al. 1981; Lowenstein 1985; which are all almost certainly related to dwar®ng. Shoshani et al. 1985b). Based on cytochrome b se- Based on the available evidence, there are two quence, Hagelberg et al. 1994) produced suggestive contradicting hypotheses on the point of origin of evidence for a closer anity of Mammuthus to Lo- the pygmy elephants of Mediterranean. The ®rst xodonta than to Elephas.HoÈ ss et al. 1994) produced supports its origin from the phylogenetic lineage sequences for a 92-bp piece of the mitochondrial 16S of mammoth, while the second from the lineage rRNA gene from four specimens of Mammuthus of Elephas. Maglio 1973) suggested that all the primigenius, but these sequence lengths were too Mediterranean pygmy elephants must be regarded short to allow a conclusion for the phylogenetic re- as derivatives of E. antiquus, while Mol et al. lationships. Yang et al. 1996, 1997a, 1997b) dem- 1996) believed that in few islands of Mediterranean onstrated the usefulness of fossil DNA sequences such as Crete and Sardinia) the pygmy elephants from the American mastodon Mammut america- must be regarded as derivatives of Mammuthus num) an extinct proboscidean, classi®ed to a dif- meridionalis. ferent family, Mammutidae) as an outgroup for Phylogenetic relationships among these genera resolving phylogeny of Elephantidae. Based on remain, however, uncertain because of lack of fossil analysis of partial cytochrome b sequences 228 bp), evidence that could provide evidence of the diver- they showed a closer clustering of two distinct in- gence process. Even though several attempts have dividuals of Mammuthus and Elephas by using the been carried out in order to resolve these relation- American mastodon sequence as an outgroup. ships, it remains a subject of controversy. Based on Ozawa et al. 1997) analyzed a longer sequence morphological studies, Elephas and Mammuthus are 1005 bases) of the mammoth cytochrome b gene considered to form a monophyletic clade within the and suggested that the mammoth is more closely subfamily Elephantinae with Loxodonta as a sister related to the Asian elephant than to the African group Shoshani et al. 1985a; Shoshani 1998). Ra- elephant. Finally, Noro et al. 1998) analyzed 366 Fig.2. Diagram showing the environmental factors which af- Tilos. Depths in meters. To the right of the absolute age scale the fected the evolution of the endemic elephants and deer of Tilos black arrows correspond to the limits of the glacials and intergla- Island. In the cave section the excavations have reached the ®rst cials aecting sea level. The main fauna of Tilos are deer strati- 8 m of sediment. Absolute dates cover the last 150,000 years in graphically older) and elephants Theodorou 1988). complete sequences of cytochrome b 1137 bases) 2). Lower down there was a layer without fossil bones and 12S rRNA 961 bases) genes in mitochondrial and after that from 4.5 m to about 7 m rare fossil DNA from the woolly mammoth Mammuthus bones from endemic deer were found Theodorou primigenius), the African elephant Loxodonta afri- 1983). cana), and Asian elephant Elephas maximus). The Originally, the two size groups were believed to results supported the hypothesis that Mammuthus is belong to two taxa, that is to Palaeoloxodon antiquus more closely related to Elephas than to Loxodonta. falconeri and Palaeoloxodon antiquus mnaidriensis. However, until now, no molecular studies have From further material Theodorou 1983) concluded included the genus of Palaeoloxodon or specimens that the two size groups corresponded to male and from the Mediterranean region. The aim of this study female animals belonging to only one taxon. Theo- is to delineate the evolutionary history of the Palae- dorou 1983) retained the name Palaeoloxodon an- oloxodon antiquus by means of DNA sequence anal- tiquus falconeri for the dwarf elephants of Tilos, ysis. Here we present cytochrome b sequence data whereas Maglio 1973) uses Elephas falconeri. from the extinct Palaeoloxodon antiquus falconeri During the last main regressive phase of the BUSK from the island of Tilos, Dodekanisa, Greece, Pleistocene 75,000±12,000 years b.p.), the sea level and use it with sequences from Mammuthus primi- was about 100 to 140 m lower than present, and the genius woolly mammoth), Mammut americanum total island area was about two to three times larger american mastodon), Elephas maximus Asiatic ele- than today, providing enough space for the immi- phant), and Loxodonta africana African elephant) to grants from the mainland, namely the elephants address long standing questions about phylogenetic Theodorou 1988). relationships of Elephantidae. During the Holocene, the equilibrium between the In 1971 Professor N. Symeonidis discovered a very elephants and the environment was very fragile. rich endemic fauna in the Charkadio cave Symeon- Three negative factors, island size reduction, man's idis 1972). Excavations removed a considerable pro- impact and volcanism, probably in that order, caused portion of sediment from the main cavern. About the extinction of these restricted endemic dwarf ele- 12,000 bones from at least 38 elephants have been phants, which became the last elephant fauna in collected. In the ®rst 3.9 m, bones belonging to two Europe to survive into the Holocene Theodorou size groups of dwarf elephants were excavated Fig. 1988). 367 Fig.3. The geographic position of Tilos Island in Dodekanisa, Greece. Materials and Methods soil to sterile polythene bags and were carried to the laboratory for further processing. Concerning the pretreatment of samples the standard aDNA handling conditions and dedicated equipment Samples were used. The island of Tilos is located north west of Rhodes in the south- east Aegean Greece). The island has an area of about 60 km2. The DNA Extraction depth of the surrounding sea corridors varies from about 900 m between Tilos and Rhodes, to 700 m between Tilos and Asia Minor Ethylenediaminetetraacetate EDTA) treatment was employed and 400 m between Nisyros and Tilos Fig.